Studies in animal models have demonstrated that calorie restriction (CR) prolongs healthy life-span. The NIH-funded Comprehensive Assessment of Long-Term Effects of Reducing Intake of Energy (CALERIE) phase 2 trial is designed to test the hypothesis that CR in humans results in similar adaptive change that occurs in animal models. The current project is an ancillary study to CALERIE which will assess an important biological mechanism by which CR may have its salutary effects on healthy aging. The ability of thymus to produce T cells that are non-reactive to self proteins but possessing a broad T cell receptor (TCR) repertoire to recognize and clear any possible foreign antigen is critical to the survival of host. The decline in cellular immunity with age is a direct consequence of progressive replacement of thymic lymphoid and stromal cell compartment with adipocytes that results in reduced generation of new T cells in a process termed as thymic involution. It is well known that TCR diversity undergoes contraction during aging due to reduction in thymopoiesis; and that this adversely impacts immune-surveillance and increases the risk of infections and cancers. It is also known that CR has immune-enhancing effects in experimental animals. We have recently identified that, ghrelin; a hunger-inducing hormone that is upregulated during CR enhances the TCR diversity via promoting the generation of naive T cells from the thymus in rodents. Therefore, we hypothesize that CR in humans will increase ghrelin production and enhance TCR diversity via promoting the generation of naove T cells from the thymus. To test this hypothesis, we propose to ascertain if the duration of 2 years of 25% CR, in well characterized CALERIE participants, is associated with a positive or unintended adverse impact on thymopoiesis. The analyses will be performed before the start of intervention at baseline, 6, 12, and 24 month in ad libitum (AL) fed and CR CALERIE subjects. The overall goal of this project is to determine if CR induced pro- thymic effects seen in animal models are reflected at cellular and molecular levels in humans. The specific Aim #1 will compare the following between CR and AL groups (a) TCR diversity, by complementarity determining region 3 length polymorphism analysis, (b) thymic output, by quantitation of T cell receptor excision circles and (c) quantitative analysis of thymic size, volume and its fat content with chemical-shift magnetic resonance imaging. Specific Aim #2 will determine if ghrelin is a primary mediator of CR's pro-thymic effects in these subjects. The data generated from this ancillary study and analyses in cooperation with the CALERIE Co-ordinating Center, will provide the first comprehensive documentation of impact of CR and the mechanisms responsible for its effects on adaptive immune system of humans. PUBLIC HEALTH RELEVANCE: Studies in animal models have demonstrated that caloric restriction without malnutrition enhances healthy life span and reduces age-related diseases. With increasing age, thymus the site of production of new T lymphocytes gets replaced with fat and looses its function. The current application will investigate if 2 years of caloric restriction in humans can stimulate the function of the thymus and thus answer key questions related to efficacy of caloric restriction as a preventive approach to strengthen the immune system in healthy non-obese people.